Compositions and methods for generating an immune response to hepatitis b virus

ABSTRACT

The compositions and methods are described for generating an immune response to a hepatitis B virus. The compositions and methods described herein relate to a modified vaccinia Ankara (MVA) vector encoding one or more viral antigens for generating a protective immune response to a hepatitis B virus, in the subject to which the vector is administered. The compositions and methods of the present invention are useful both prophylactically and therapeutically and may be used to prevent and/or treat an infection caused by hepatitis B virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/305,305, filed on Nov. 28, 2018, which is a national stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2017/034983, filed May 30, 2017, which claims the benefit of U.S.Provisional Application No. 62/343,074, filed May 30, 2016, each ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to compositions, including vaccinecompositions, for generating an immune response to a hepatitis B virusin a subject to which the composition is administered, as well asmethods of manufacture and use of such compositions. More specifically,the compositions and methods described herein relate to a modifiedvaccinia Ankara (MVA) vector encoding one or more viral antigens,suitable for use in generating a protective immune response to ahepatitis B virus in a subject to which the vector is administered. Thecompositions and methods of the present invention are useful bothprophylactically and therapeutically.

INCORPORATION BY REFERENCE

The contents of the text file named“19101-017US2_2021-07-06_Seq_Listing_ST25.txt” which was created on Jul.6, 2021 and is 11.4 KB in size, are hereby incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

Despite great progress in antiviral treatments, hepatitis B virus (HBV)infection is still a major global public health problem. Approximately 2billion people have been infected worldwide during their lifetime, andmore than 350 million are chronic carriers of the virus (Liaw Y F, etal. Lancet 2009, 373:582-592). HBV is not cytopathic per se, but thehost antiviral immune response to envelope, capsid and Pol proteinsresults in hepatocyte damage. Specifically, CD4⁺ and CD8⁺ T-cellresponses have been shown to play a central role in the outcome ofinfection. (Bauer T et al., Dig. Dis. 2011; 29:423-433) Various studieshave shown that CD4⁺ helper T-cell- and CD8⁺ cytotoxic T-cell-mediatedimmune responses determine the outcome of HBV infection. Thus,spontaneous viral clearance of HBV infection is characterized byvigorous and sustained multi-epitope-specific CD4⁺ and CD8⁺ T-cellresponses during the acute phase of infection. In contrast, chronicinfection with HBV is correlated with late, transient, weak or narrowlyfocused CD4⁺ and CD8⁺ T-cell responses. (Ferrari C. et al., J Immunol.1990; 145:3442-3449; Rehermann B. et al., J Exp Med. 1995;181:1047-1058) However, it is important to note that the effects of CD4⁺and CD8⁺ T-cell responses are not only important for viral control butalso implicated in liver injury and the establishment of liver diseasesin HBV infections. (Maini M K, et al., J Exp Med. 2000; 191:1269-1280)HBV infection may cause acute and chronic hepatitis, which leads toliver cirrhosis (LC) and hepatocellular carcinoma (HCC) (Chu C M. JGastroenterol Hepatol 2000 15 Suppl:E25-30).

Not all available HBV vaccines are broadly effective. Current HBVvaccines on the market, which protect most people prophylacticallyagainst HBV infection contain only the S. antigen. Almost 5 to 10%people vaccinated prophylactically with the available vaccines fail tomount an adequate antibody response to offer protection (Kubba A K, etal. Commun Dis Public Health 2003 6: 106-112). Furthermore, no HBVvaccine currently available is effective therapeutically. Once an HBVinfection becomes established as a chronic infection, mounting aneffective immune response against the virus becomes still more difficultbecause the immune system grows tolerant to the persisting virus.

What is needed is a vaccine or immune response stimulating compositionto break tolerance to the hepatitis B surface antigen (HBsAg/Australiaantigen) and other HBV antigens, to induce anti-HBsAg neutralizingantibodies, and to induce productive CD4⁺ and CD8⁺ T cell responses.

SUMMARY OF THE INVENTION

The compositions and methods of the invention described herein areuseful for generating an immune response to at least one hepatitis Bvirus in a subject in need thereof. Advantageously, the compositions andmethods may be used prophylactically to immunize a subject against ahepatitis B virus infection, or used therapeutically to prevent, treator ameliorate the onset and severity of disease.

In a first aspect, the present invention is a recombinant modifiedvaccinia Ankara (MVA) vector comprising one or more nucleic acidsequence encoding a hepatitis B virus polypeptide or fusion protein,wherein the at least one nucleic acid sequence is inserted into the MVAvector under the control of at least one promoter compatible with apoxvirus expression system.

In one embodiment, the recombinant MVA vector comprises two or morenucleic acid sequences encoding hepatitis B virus proteins, wherein theat least two nucleic sequences are inserted into the MVA vector underthe control of at least two promoters capable compatible with poxvirusexpression systems.

In one embodiment, the recombinant MVA vector comprises a first nucleicacid sequence encoding one or more hepatitis structural proteins and asecond nucleic sequence encoding one or more hepatitis B nonstructuralproteins, wherein both the first and second nucleic acid sequences areinserted into the MVA vector under the control of promoters compatiblewith poxvirus expression systems.

In one embodiment, the hepatitis B virus structural protein comprisesPreS2-S protein or fragments thereof.

In some embodiments, the hepatitis B virus structural protein comprisesa fragment of the PreS2-S protein lacking all or part of the S domain.

In one embodiment, the hepatitis B virus structural protein is a fusionprotein.

In one embodiment, the hepatitis B virus structural protein is a preS.HAfusion protein. In one embodiment, the hepatitis B virus non-structuralprotein are selected from PreC-C, and truncated X protein, and fragmentsthereof.

In one embodiment, the hepatitis B virus non-structural protein areselected from PreC-C, and X protein, and fragments thereof.

In one embodiment, the hepatitis B virus non-structural protein is afusion protein.

In one embodiment, the hepatitis B virus non-structural protein isM1.P41A.

In one embodiment, the first and second nucleic acid sequences areinserted into one or more deletion sites of the recombinant MVA vector.

In one embodiment, the first and second nucleic acid sequences areinserted into the recombinant MVA vector in a natural deletion site, amodified natural deletion site, or between essential or non-essentialMVA genes.

In another embodiment, the first and second nucleic acid sequences areinserted into the same natural deletion site, a modified naturaldeletion site, or between the same essential or non-essential MVA genes.

In another embodiment, the first nucleic acid sequence is inserted intoa deletion site selected from I, II, III, IV, V or VI and thenonstructural protein sequence is inserted into a deletion site selectedfrom I, II, III, IV, V or VI.

In another embodiment, the first nucleic sequence is inserted in a firstdeletion site and the second nucleic acid sequence is inserted into asecond deletion site.

In a particular embodiment, the first nucleic acid sequence is insertedbetween two essential and highly conserved MVA genes and the secondnucleic acid sequence is inserted into a restructured and modifieddeletion site III.

In a particular embodiment, the first nucleic acid sequence is insertedbetween two essential and highly conserved MVA genes to limit theformation of viable deletion mutants.

In a particular embodiment, the first nucleic acid sequence is insertedbetween MVA genes, I8R and G1L.

In a particular embodiment, the first nucleic acid sequence is insertedbetween MVA genes, I8R and G1L and the second nucleic acid sequence isinserted into modified deletion site III.

In one embodiment, the promoter is selected from the group consisting ofPm2H5, PsynII, mH5 promoters, or combinations thereof.

In one embodiment, the recombinant MVA vector expresses one or morestructural proteins and non-structural proteins that assemble into VLPs.

In one embodiment, the structural protein sequence and thenon-structural protein sequence are from a hepatitis B genotype A, B, C,D, E, F, G, or H.

In one embodiment, the structural protein sequence and thenon-structural protein sequence are from a hepatitis B genotype D.

In a second aspect, the present invention is a pharmaceuticalcomposition comprising the recombinant MVA vector of the presentinvention and a pharmaceutically acceptable carrier.

In one embodiment, the recombinant MVA vector is formulated forintraperitoneal, intramuscular, intradermal, epidermal, mucosal orintravenous administration.

In one embodiment, the recombinant MVA vector is formulated forintramuscular administration.

In a third aspect, the present invention is a pharmaceutical compositioncomprising a first recombinant MVA vector and a second recombinant MVAvector, each comprising a first nucleic acid sequence encoding ahepatitis B virus structural protein and a second nucleic acid sequenceencoding a hepatitis B virus non-structural protein, wherein (i) thefirst nucleic acid sequence of the first recombinant MVA vector isdifferent than the first nucleic acid sequence of the second recombinantMVA vector and/or (ii) the second nucleic acid sequence of the firstrecombinant MVA vector is different than the second nucleic acidsequence of the second recombinant MVA vector.

In a particular embodiment, the first nucleic sequence of the first andsecond recombinant MVA vector encodes PreS2_S or PreS.HA, and the firstnucleic acid sequence of the first recombinant MVA vector is from thesame or a different genotype than the first nucleic acid sequence of thesecond recombinant MVA vector.

In one embodiment, the first and second sequences of the firstrecombinant MVA vector are from genotype B and the first and secondsequences of the second recombinant MVA vector are from genotype C.

In one embodiment, the first and second sequences of the firstrecombinant MVA vector are from genotype A and the first and secondsequences of the second recombinant MVA vector are from genotype D.

In one embodiment, the first and second sequences of the firstrecombinant MVA vector are from genotype C and the first and secondsequences of the second recombinant MVA vector are from genotype D.

In one embodiment, the pharmaceutical composition comprises fourrecombinant MVA vectors where the first and second sequences of each ofthe four vectors are from genotypes A, B, C, and D respectively.

In another particular embodiment, the second nucleic acid sequence ofthe first recombinant MVA vector is from a different genotype than thesecond nucleic acid sequence of the second recombinant MVA vector.

In various embodiments, the first nucleic acids sequences encodingstructural proteins are selected from genotypes A, B, C, or D and thesecond nucleic acid sequences encoding nonstructural proteins areselected from genotypes C and D.

In a particular embodiment, the first nucleic acid sequence of eachrecombinant vector are from the same genotype.

In a fifth aspect, the present invention is a method of inducing animmune response in a subject in need thereof, said method comprisingadministering the composition of the present invention to the subject inan amount sufficient to induce an immune response.

In one embodiment, the composition is administered prophylactically toimmunize a subject against hepatitis B virus infection.

In one embodiment, the composition is administered therapeutically toprevent, treat or ameliorate the onset and severity of disease.

In one embodiment, the immune response is a humeral immune response, acellular immune response or a combination thereof.

In a particular embodiment, the immune response comprises production ofbinding antibodies against the hepatitis B virus.

In a particular embodiment, the immune response comprises production ofneutralizing antibodies against the hepatitis B virus.

In a particular embodiment, the immune response comprises production ofnon-neutralizing antibodies against the hepatitis B virus.

In a particular embodiment, the immune response comprises production ofa cell-mediated immune response against the hepatitis B virus.

In a particular embodiment, the immune response comprises production ofa CD8⁺ T cell response against the hepatitis B virus.

In a particular embodiment, the immune response comprises production ofneutralizing and non-neutralizing antibodies against the hepatitis Bvirus.

In a particular embodiment, the immune response comprises production ofneutralizing antibodies and cell-mediated immunity against the hepatitisB virus.

In a particular embodiment, the immune response comprises production ofnon-neutralizing antibodies and cell-mediated immunity against thehepatitis B virus.

In a particular embodiment, the immune response comprises production ofneutralizing antibodies, non-neutralizing antibodies, and cell-mediatedimmunity against the hepatitis B virus.

In a particular embodiment, the immune response comprises production ofneutralizing antibodies and CD8⁺ T cell immunity against the hepatitis Bvirus.

In a particular embodiment, the immune response comprises production ofnon-neutralizing antibodies and CD8⁺ T cell immunity against thehepatitis B virus.

In a particular embodiment, the immune response comprises production ofneutralizing antibodies, non-neutralizing antibodies, and CD8⁺ T cellimmunity against the hepatitis B virus.

In one embodiment, the immune response is considered a surrogate markerfor protection against a hepatitis B virus.

In one embodiment, the method induces an immune response against ahepatitis B virus.

In a sixth aspect, the present invention is a method of preventing ahepatitis B virus infection in a subject in need thereof, said methodcomprising administering the recombinant MVA vector of the presentinvention to the subject in a prophylactically effective amount.

In an seventh aspect, the present invention is a method of treatinghepatitis B virus infection in a subject in need thereof, said methodcomprising administering the recombinant MVA vector in a therapeuticallyeffective amount to the subject.

In another embodiment, the method results in reduction or elimination ofthe subject's ability to transmit the infection to a subject.

In one embodiment, the method prevents or ameliorates a hepatitis Bvirus infection.

In an eighth aspect, the present invention is a method manufacturing arecombinant MVA vector comprising inserting at least one nucleic acidsequence encoding PreS2_S or PreS.HA and at least one nucleic acidsequence encoding a non-structural protein sequence into the recombinantMVA vector, wherein each nucleic acid sequence is operably linked to apromoter compatible with a poxvirus expression system.

In one embodiment, the non-structural sequence is PreCore/Core,truncated X gene or M1.P41A.

In one embodiment, the recombinant MVA viral vector expresses hepatitisB virus PreS2_S and PreCore/Core and Truncated X proteins that assembleinto VLPs.

In one embodiment, the recombinant MVA viral vector expresses hepatitisB virus PreS2_S and M1.P41A proteins that assemble into VLPs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic for the shuttle vector for hepatitis B virusPreS2_S.

The ampicillin resistance marker, allowing the vector to replicate inbacteria is illustrated with a block labeled “amp-R.” The two flankingsequences, allowing the vector to recombine with the MVA genome, areillustrated with a block labeled “Flank 1” and a block labeled “Flank 2”respectively. The green fluorescent protein (GFP) selection marker,allowing the selection of recombinant MVAs, is illustrated with an arrowlabeled “GFP.” The block labeled “DR” illustrates the location of asequence homologous to part of Flank 1 of the MVA sequence. DR enablesremoval of the GFP sequence from the MVA vector after insertion ofPreS2_S into the MVA genome. The modified H5 (mH5) promoter, whichenables transcription of the inserted heterologous gene, is illustratedwith a triangle between the DR and PreS2_S elements. The hepatitis Bvirus PreS2_S gene is illustrated with an arrow labeled “PreS2_S”.

FIG. 2 is a schematic for the shuttle vector for hepatitis virusPreCore/Core tr.X.

The ampicillin resistance marker, allowing the vector to replicate inbacteria, is illustrated with a block labeled “amp-R.” The two flankingsequences, allowing the vector to recombine with the MVA genome, areillustrated with blocks labeled “A50R” and “B1R”. The green fluorescentprotein (GFP) selection marker, allowing the selection of recombinantMVAs, is illustrated with an arrow labeled “GFP.” The block labeled “DR”illustrates the location of a sequence homologous to part of A50R of theMVA sequence. DR enables removal of the GFP sequence from the MVA vectorafter insertion of sequences into the MVA genome. The modified vacciniavirus P7.5 promoter, which enables transcription of the insertedheterologous gene, is illustrated with a triangle between the DR andPreCore/Core elements. The hepatitis B PreCore/Core gene is illustratedwith an arrow labeled “PreCore-Core.” The hepatitis B truncated X geneis illustrated with an arrow labeled “trc. X.”

FIG. 3 is a schematic for the shuttle vector for hepatitis B fusionprotein preS.HA.

The ampicillin resistance marker, allowing the vector to replicate inbacteria, is illustrated with a block labeled “amp-R.” The two flankingsequences, allowing the vector to recombine with the MVA genome, areillustrated with blocks labeled “Flank 1” and “Flank 2.” The greenfluorescent protein (GFP) selection marker, allowing the selection ofrecombinant MVAs, is illustrated with an arrow labeled “GFP.” The blocklabeled “DR” illustrates the location of a sequence homologous to partof Flank 1 of the MVA sequence. DR enables removal of the GFP sequencefrom the MVA vector after insertion of PreS.HA into the MVA genome. Themodified H5 (mH5) promoter, which enables transcription of the insertedheterologous gene, is illustrated with a triangle between the DR andPreS.HA elements. The hepatitis B fusion protein PreS.HA gene isillustrated with an arrow labeled “PreS.HA.”

FIG. 4 is a schematic for the shuttle vector for hepatitis B fusionprotein M1.P41A.

The ampicillin resistance marker, allowing the vector to replicate inbacteria, is illustrated with a block labeled “amp-R.” The two flankingsequences, allowing the vector to recombine with the MVA genome, areillustrated with blocks labeled “A50R” and “B1R”. The green fluorescentprotein (GFP) selection marker, allowing the selection of recombinantMVAs, is illustrated with an arrow labeled “GFP.” The block labeled “DR”illustrates the location of a sequence homologous to part of A50R of theMVA sequence. DR enables removal of the GFP sequence from the MVA vectorafter insertion of M1.P4A into the MVA genome. The modified H5 (mH5)promoter, which enables transcription of the inserted heterologous gene,is illustrated with a triangle between the DR and M1.P4A elements. Thehepatitis B fusion protein M1.P4A gene is illustrated with an arrowlabeled “M1.P4A.”

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods are provided to produce an immune response to ahepatitis B virus, in a subject in need thereof. The compositions andmethods of the present invention can be used to prevent infection in anunexposed person or to treat disease in a subject exposed to a hepatitisB virus who is not yet symptomatic or has minimal symptoms, or to treatdisease in a subject with active chronic hepatitis B virus infection. Inone embodiment, treatment limits an infection and/or the severity ofdisease.

Ideal immunogenic compositions or vaccines are safe, effective, andprovide sufficient scope of protection and longevity. However,compositions having fewer than all of these characteristics may still beuseful in preventing viral infection or limiting symptoms or diseaseprogression in an exposed subject treated prior to the development ofsymptoms or limiting symptoms or disease progression in an exposedsubject treated after to the development of symptoms. In one embodimentthe present invention provides a vaccine that permits at least partial,if not complete, protection after a single immunization.

In exemplary embodiments, the immune responses are long-lasting anddurable so that repeated boosters are not required, but in oneembodiment, one or more administrations of the compositions providedherein are provided to boost the initial primed immune response.

I. Definitions

Where a term is provided in the singular, the inventors also contemplateaspects of the invention described by the plural of that term. As usedin this specification and in the appended claims, the singular forms“a”, “an” and “the” include plural references unless the context clearlydictates otherwise, e.g., “a peptide” includes a plurality of peptides.Thus, for example, a reference to “a method” includes one or moremethods, and/or steps of the type described herein and/or which willbecome apparent to those persons skilled in the art upon reading thisdisclosure.

The term “antigen” refers to a substance or molecule, such as a protein,or fragment thereof, that is capable of being a target of an immuneresponse.

The term “binding antibody” or “bAb” refers to an antibody which eitheris purified from, or is present in, a body fluid (e.g., serum or amucosal secretion) and which recognizes a specific antigen. As usedherein, the antibody can be a single antibody or a plurality ofantibodies. Binding antibodies comprise neutralizing andnon-neutralizing antibodies.

The term “cell-mediated immune response” refers to the immunologicaldefense provided by lymphocytes, such as the defense provided bysensitized T cell lymphocytes when they directly lyse cells expressingforeign antigens and secrete cytokines (e.g., IFN-gamma.), which canmodulate macrophage and natural killer (NK) cell effector functions andaugment T cell expansion and differentiation. The cellular immuneresponse is one of two branches of the adaptive immune response.

The term “conservative amino acid substitution” refers to substitutionof a native amino acid residue with a non-native residue such that thereis little or no effect on the size, polarity, charge, hydrophobicity, orhydrophilicity of the amino acid residue at that position, and withoutresulting in substantially altered immunogenicity. For example, thesemay be substitutions within the following groups: valine, glycine;glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamicacid; asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. Conservative amino acid modifications to thesequence of a polypeptide (and the corresponding modifications to theencoding nucleotides) may produce polypeptides having functional andchemical characteristics similar to those of a parental polypeptide.

The terms “gene”, “polynucleotide”, “nucleotide” and “nucleic acid” areused interchangeably herein.

The term “fragment” in the context of a proteinaceous agent refers to apeptide or polypeptide comprising an amino acid sequence of at least 2contiguous amino acid residues, at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least 100 contiguous aminoacid residues, at least 125 contiguous amino acid residues, at least 150contiguous amino acid residues, at least 175 contiguous amino acidresidues, at least 200 contiguous amino acid residues, or at least 250contiguous amino acid residues of the amino acid sequence of a peptide,polypeptide or protein.

In one embodiment, a fragment of a full-length protein retains activityof the full-length protein. In another embodiment, the fragment of thefull-length protein does not retain the activity of the full-lengthprotein.

The term “fragment” in the context of a nucleic acid refers to a nucleicacid comprising an nucleic acid sequence of at least 2 contiguousnucleotides, at least 5 contiguous nucleotides, at least 10 contiguousnucleotides, at least 15 contiguous nucleotides, at least 20 contiguousnucleotides, at least 25 contiguous nucleotides, at least 30 contiguousnucleotides, at least 35 contiguous nucleotides, at least 40 contiguousnucleotides, at least 50 contiguous nucleotides, at least 60 contiguousnucleotides, at least 70 contiguous nucleotides, at least contiguous 80nucleotides, at least 90 contiguous nucleotides, at least 100 contiguousnucleotides, at least 125 contiguous nucleotides, at least 150contiguous nucleotides, at least 175 contiguous nucleotides, at least200 contiguous nucleotides, at least 250 contiguous nucleotides, atleast 300 contiguous nucleotides, at least 350 contiguous nucleotides,or at least 380 contiguous nucleotides of the nucleic acid sequenceencoding a peptide, polypeptide or protein. In a preferred embodiment, afragment of a nucleic acid encodes a peptide or polypeptide that retainsactivity of the full-length protein. In another embodiment, the fragmentencodes a peptide or polypeptide that of the full-length protein doesnot retain the activity of the full-length protein. As used herein, theterm “hepatitis B genotype” refers to difference classifications ofhepatitis B virus differentiated into many genotypes, according togenome sequence. There are eight well-known genotypes (A, B, C, D, E, F,G, H, I and J) of the HBV genome that have been identified. Genotypes Cand D are recognized as being causative of more severe disease.

As used herein, the phrase “hepatitis B polypeptide” refers to anyhepatitis B polypeptide or fusion protein described herein for use ingenerating an immune response to hepatitis B including structuralpolypeptides PreS2_S and PreS.HA and nonstructural polypeptidesPreCore/Core, or truncated X protein or M1.P41A.

As used herein, the phrase “heterologous sequence” refers to any nucleicacid, protein, polypeptide or peptide sequence which is not normallyassociated in nature with another nucleic acid or protein, polypeptideor peptide sequence of interest.

As used herein, the phrase “heterologous gene insert” refers to anynucleic acid sequence that has been, or is to be inserted into therecombinant vectors described herein. The heterologous gene insert mayrefer to only the gene product encoding sequence or may refer to asequence comprising a promoter, a gene product encoding sequence (suchas GP, VP or Z), and any regulatory sequences associated or operablylinked therewith.

The term “homopolymer stretch” refers to a sequence comprising at leastfour of the same nucleotides uninterrupted by any other nucleotide,e.g., GGGG or TTTTTTT.

The term “humeral immune response” refers to the stimulation of antibody(Ab) production. Humeral immune response also refers to the accessoryproteins and events that accompany Ab production, including T helpercell activation and cytokine production, affinity maturation, and memorycell generation. The humeral immune response is one of two branches ofthe adaptive immune response.

The term “humeral immunity” refers to the immunological defense providedby antibody, such as neutralizing Ab that can directly block infection;or, binding Ab that identifies a virus or infected cell for killing bysuch innate immune responses as complement (C′)-mediated lysis,phagocytosis, and natural killer cells.

The term “immune” or “immunity” refers to protection from disease (e.g.,preventing or attenuating (e.g., suppression) of a sign, symptom orcondition of the disease) upon exposure to a pathogen (e.g., a virus)capable of causing the disease.

The term “immune response” refers to any response to an antigen orantigenic determinant by the immune system of a subject (e.g., a human).Exemplary immune responses include humeral immune responses (e.g.,production of antigen-specific antibodies) and cell-mediated immuneresponses (e.g., production of antigen-specific T cells).

The term “immunogen” refers to a substance or molecule, such as a virus,a protein, or fragment thereof, that can induce an immune response.

The term “immunogenic” refers to the capability of a substance ormolecule, such as a virus, a protein, or fragment thereof, to induce animmune response.

The term “improved therapeutic outcome” relative to a subject diagnosedas infected with a particular virus (e.g., a hepatitis B virus) refersto a slowing or diminution in the growth of virus, or viral load, ordetectable symptoms associated with infection by that particular virus;or a reduction in the ability of the infected subject to transmit theinfection to another, uninfected subject.

The term “inducing an immune response” means eliciting a humeralresponse (e.g., the production of antibodies) or a cellular response(e.g., the activation of T cells) directed against a virus (e.g.,hepatitis B virus) or other immunogen in a subject to which thecomposition (e.g., a vaccine) has been administered.

The term “insertion” in the context of a polypeptide or protein refersto the addition of one or more non-native amino acid residues in thepolypeptide or protein sequence. Typically, no more than about from 1 to6 residues (e.g., 1 to 4 residues) are inserted at any one site withinthe polypeptide or protein molecule. The term “insertion” in the contextof a polynucleotide or nucleic acid refers to the addition of one ormore non-native nucleic acid residues in the polynucleotide or nucleicacid sequence. Typically, no more than about from 1 to 10,000 residuesare inserted at any one site within the polynucleotide or nucleic acidmolecule.

The term “modified vaccinia Ankara,” “modified vaccinia ankara,”“Modified Vaccinia Ankara,” or “MVA” refers to a highly-attenuatedstrain of vaccinia virus developed by Dr. Anton Mayr by serial passageon chick embryo fibroblast cells; or variants or derivatives thereof.MVA is reviewed in (Mayr, A. et al. 1975 Infection 3:6-14; Swiss PatentNo. 568,392).

The term “neutralizing antibody” or “NAb” refers to an antibody which iseither purified from, or is present in, a body fluid (e.g., serum or amucosal secretion) and which recognizes a specific antigen and inhibitsthe effect(s) of the antigen in the subject (e.g., a human). As usedherein, the antibody can be a single antibody or a plurality ofantibodies.

The term “non-neutralizing antibody” or “nnAb” refers to a bindingantibody that is not a neutralizing antibody.

The term “operably linked”, when used with reference to a promoter,refers to a configuration in which the promoter is placed at anappropriate position relative to the coding sequence of a polynucleotidesuch that the promoter directs expression of the coding sequence.

The term “prevent”, “preventing” and “prevention” refers to theinhibition of the development or onset of a condition (e.g., a hepatitisB infection or a condition associated therewith), or the prevention ofthe recurrence, onset, or development of one or more symptoms of acondition in a subject resulting from the administration of a therapy orthe administration of a combination of therapies.

The term “prophylactically effective amount” refers to the amount of acomposition (e.g., the recombinant MVA vector or pharmaceuticalcomposition) which is sufficient to result in the prevention of thedevelopment, recurrence, or onset of a condition or a symptom thereof(e.g., a hepatitis B infection or a condition or symptom associatedtherewith) or to enhance or improve the prophylactic effect(s) ofanother therapy.

The term “recombinant” means a polynucleotide of semisynthetic, orsynthetic origin that either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

The term “recombinant,” with respect to a viral vector, means a vector(e.g., a viral genome) that has been manipulated in vitro (e.g., usingrecombinant nucleic acid techniques) to express heterologous viralnucleic acid sequences.

The term “regulatory sequence” or “regulatory sequences” referscollectively to promoter sequences, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, internal ribosome entry sites (“IRES”),enhancers, and the like, which collectively provide for thetranscription and translation of a coding sequence. Not all of thesecontrol sequences need always be present so long as the selected gene iscapable of being transcribed and translated.

The term “shuttle vector” refers to a genetic vector (e.g., a DNAplasmid) that is useful for transferring genetic material from one hostsystem into another. A shuttle vector can replicate alone (without thepresence of any other vector) in at least one host (e.g., E. coli). Inthe context of MVA vector construction, shuttle vectors are usually DNAplasmids that can be manipulated in E. coli and then introduced intocultured cells infected with MVA vectors, resulting in the generation ofnew recombinant MVA vectors.

The term “silent mutation” means a change in a nucleotide sequence thatdoes not cause a change in the primary structure of the protein encodedby the nucleotide sequence, e.g., a change from AAA (encoding lysine) toAAG (also encoding lysine).

The term “subject” means any mammal, including but not limited to,humans, domestic and farm animals, and zoo, sports, or pet animals, suchas dogs, horses, cats, cows, rats, mice, guinea pigs and the like.

The term “surrogate endpoint” means a clinical measurement other than ameasurement of clinical benefit that is used as a substitute for ameasurement of clinical benefit.

The term “surrogate marker” means a laboratory measurement or physicalsign that is used in a clinical or animal trial as a substitute for aclinically meaningful endpoint that is a direct measure of how a subjectfeels, functions, or survives and is expected to predict the effect ofthe therapy (Katz, R., NeuroRx 1:189-195 (2004); New drug, antibiotic,and biological drug product regulations; accelerated approval-FDA. Finalrule. Fed Regist 57: 58942-58960,1992.).

The term “surrogate marker for protection” means a surrogate marker thatis used in a clinical or animal trial as a substitute for the clinicallymeaningful endpoint of prevention of hepatitis B virus infection.

The term “synonymous codon” refers to the use of a codon with adifferent nucleic acid sequence to encode the same amino acid, e.g., AAAand AAG (both of which encode lysine). Codon optimization changes thecodons for a protein to the synonymous codons that are most frequentlyused by a vector or a host cell.

The term “therapeutically effective amount” means the amount of thecomposition (e.g., the recombinant MVA vector or pharmaceuticalcomposition) that, when administered to a mammal for treating aninfection, is sufficient to effect treatment for the infection.

The term “treating” or “treat” refer to the eradication or control of ahepatitis B virus, a reduction in the titer of the hepatitis B virus, areduction in the numbers of the hepatitis B virus, the reduction oramelioration of the progression, severity, and/or duration of acondition or one or more symptoms caused by the hepatitis B virusresulting from the administration of one or more therapies, or thereduction or elimination of the subject's ability to transmit theinfection to another uninfected subject.

The term “vaccine” means material used to provoke an immune response andconfer immunity after administration of the material to a subject. Suchimmunity may include a cellular or humeral immune response that occurswhen the subject is exposed to the immunogen after vaccineadministration.

The term “vaccine insert” refers to a nucleic acid sequence encoding aheterologous sequence that is operably linked to a promoter forexpression when inserted into a recombinant vector. The heterologoussequence may encode a hepatitis B protein described here.

The term “viral infection” means an infection by a viral pathogen (e.g.,a hepatitis B virus) wherein there is clinical evidence of the infectionbased on symptoms or based on the demonstration of the presence of theviral pathogen in a biological sample from the subject.

The term “virus-like particles” or “VLP” refers to a structure whichresembles the native virus antigenically and morphologically.

II. Hepatitis B Virus Genotypes and Sequences

The compositions of the present invention are useful for inducing animmune response to a hepatitis B virus.

There are 10 identified genotypes of hepatitis B virus (genotypes A, B,C, D, E, F, G, H, I, and J).

In one embodiment, sequences corresponding to genotypes C or D areemployed in the MVA vectors described herein. Genotypes C and Darerecognized as causing more severe disease.

In one embodiment, sequences corresponding to genotype D are employed inthe MVAvectors described herein.

In another embodiment, sequences corresponding to genotypes B and C areemployed in the MVA vectors described herein.

In another embodiment, sequences corresponding to genotypes A and D areemployed in the MVA vectors described herein.

In another embodiment, sequences corresponding to genotypes A, B, C, andD are employed in the MVA vectors described herein.

There are four known genes encoded by the genome called C, P, S, and X.The core protein is coded for by gene C (HBcAg), and its start codon ispreceded by an upstream in-frame AUG start codon from which the pre-coreprotein is produced. HBcAg is produced by proteolytic processing of thepre-core protein. The DNA polymerase is encoded by gene P. Gene S is thegene that codes the surface antigen (HBsAg). The HBsAg gene is one longopen reading frame but contains three in frame “start” (ATG) codons thatdivide the gene into three sections, pre-S1, pre-S2, and S. Because ofthe multiple start codons, polypeptides of three different sizes calledlarge, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) are produced.The function of the protein coded for by gene X is not fully understood,but some evidence suggests that it may function as a transcriptionaltransactivator.

A. Antigen Sequences Used in MVA Vectors

In one embodiment, the MVA vector expresses a polypeptide comprising ahepatitis B preS2_S epitope, or an antigenic fragment thereof, canprovide Band T cell epitopes that promote the humeral and cellularresponses and enhance the seroprotection rate by overcomingnon-responsiveness to the S antigen-only vaccines. Therefore,compositions and methods are disclosed using a preS antigen to developvaccines and immune therapies for treating or preventing hepatitis Binfection.

In some embodiments, a preS2_S sequence is incorporated into an MVAvector, which when expressed produce virus-like particle (VLP) that canbe used, for example, as a vaccine.

In some embodiments, a preCore/Core sequence is incorporated into an MVAvector, which when expressed produce VLP that can be used, for example,as a vaccine that elicits Ab responses or T cell responses or Ab and Tcell responses.

In some embodiments, a truncated X sequence is incorporated into an MVAvector, which when expressed produces truncated X protein that can beused, for example, as a vaccine that elicits Ab responses or T cellresponses or Ab and T cell responses.

In some embodiments, a PreS.HA and a M1.P41A sequence is incorporatedinto an MVA vector, which when expressed produce VLP that can be used,for example, as a vaccine that elicits Ab responses or T cell responsesor Ab and T cell responses.

Antigen epitopes Sequence PreS2-S (PreS 55AA + S 212AA) SEQ ID N0: 1PreC-C (PreC 29AA + C 183AA) SEQ ID N0: 2 Truncated X protein (includingSEQ ID N0: 3 MHCI and MHCII epitopes) PreS.HA SEQ ID N0: 4 M1.P41A SEQID N0: 5

B. Hepatitis B preS.HA Fusion Protein

In some embodiments, the hepatitis B virus structural protein comprisesa fragment of the PreS2-S protein lacking all or part of the S domain(i.e., the preS antigen). The S domain can therefore be replaced with analternative transmembrane domain in some embodiments. For example, afusion protein is disclosed that comprises a hepatitis B preS antigenfused at the N-terminus to a transmembrane domain and optionalcytoplasmic tail of a viral envelope protein. Viral envelope proteinsthat contain transmembrane domains suitable for VLP formation includeinfluenza virus hemagglutinin (HA) protein, a type I transmembraneprotein. The hepatitis B preS antigen may also be fused with other typeI transmembrane glycoproteins, such as glycoproteins from arenaviruses,bunyaviruses, coronaviruses, filoviruses, paramyxoviruses, retroviruses,and togaviruses.

In one embodiment, the MVA vector expresses a fusion protein comprisinga hepatitis B preS epitope fused to the HA protein, or an antigenicfragment thereof, can provide B and T cell epitopes that promote thehumeral and cellular responses and enhance the seroprotection rate byovercoming non-responsiveness to the S antigen-only vaccines. Therefore,compositions and methods are disclosed using a preS antigen to developvaccines and immune therapies for treating or preventing hepatitis Binfection.

In some embodiments, a preS fusion antigen is incorporated into avirus-like particle (VLP) that can be used, for example, as a vaccine orto active T cells. In one embodiment, the preS antigen can beincorporated into a fusion protein that will incorporate into a VLP. Forexample, a fusion protein is disclosed that comprises a hepatitis B preSantigen fused at the N-terminus to a transmembrane domain and optionalcytoplasmic tail of a viral envelope protein.

In one embodiment, the expressed hepatitis B preS antigen has the aminoacid sequence: MGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAF HQALQDPRVRGL YLPAG GSSSGTVNPA PNIASHISSISARTGDPVTN (SEQ ID NO: 6), or aconservative variant thereof having at least about 70%, 80%, or 90%sequence identity to SEQ ID NO: 6 (i.e., one, two, or three conservativeamino acid substitutions).

In some embodiments, the disclosed HBVpreS.HA (SHA) fusion proteincorresponding to the nucleotide sequence of SEQ ID NO:4 has the aminoacid sequence: MEAKLFVLFC AFT ALKAMGT NLSVPNPLGF FPDHQLDP AFGANSNNPDWDFNPIKDHWPA ANQVGVGAFG PGL TPPHGGI LGWSPQAQGILTTVSTIPPPASTNRQSGRQ PTPISPPLRD SHPQAMQWNS TAFHQALQDP RVRGLYLPAGGSSSGTVNPA PNIASHISSI SARTGDPVTN KLESVGVHQI LAIYSTVASSL VLL VSLGAISFWMCSNGSL QCRICI (SEQ ID NO:7), or a conservative variant thereofhaving at least about 70%, 80%, or 90% sequence identity to SEQ ID NO:7(i.e., one, two, or three conservative amino acid substitutions).

In some embodiments, the influenza virus M1.P41A protein has the aminoacid sequence corresponding to the translated nucleotide sequence of SEQID NO:5: MSLL TEVETY VLSIIPSGPL KAEIAQRLEG VF AGKNTDLEALMEWLKTRPILSPLTKGIL GFVFTLTVPS ERGLQRRRFV QNALNGNGDPNNMDRA VKL Y KKLKREITFHGAKEVSLSYS TGALASCMGL IYNRMGTVTTEAAFGLVCAT CEQIADSQHR SHRQMATTTNPLIRHENRMV LASTTAKAM EQMAGSSEQAAEAMEVASQTRQMVHAMRTIGTHPSSSAGLKDDLLENLQAYQK RMGVQIQRFK (SEQ ID NO:8), or a conservativevariant thereof having at least about 70%, 80%, or 90% sequence identityto SEQ ID NO:8 (i.e., one, two, or three conservative amino acidsubstitutions).

III. Recombinant Viral Vectors

In one aspect, the present invention is a recombinant viral vectorcomprising one or more genes of a hepatitis B virus. In certainembodiments, the recombinant viral vector is a vaccinia viral vector,and more particularly, an MVA vector, comprising one or more genes ofahepatitis B virus.

Vaccinia viruses have also been used to engineer viral vectors forrecombinant gene expression and for the potential use as recombinantlive vaccines (Mackett, M. et al 1982 PNAS USA 79:7415-7419; Smith, G.L. et al. 1984 Biotech Genet Engin Rev 2:383-407). This entails DNAsequences (genes) which code for foreign antigens being introduced, withthe aid of DNA recombination techniques, into the genome of the vacciniaviruses. If the gene is integrated at a site in the viral DNA which isnon-essential for the life cycle of the virus, it is possible for thenewly produced recombinant vaccinia virus to be infectious, that is tosay able to infect foreign cells and thus to express the integrated DNAsequence (EP Patent Applications No. 83,286 and No. 110,385). Therecombinant vaccinia viruses prepared in this way can be used, on theone hand, as live vaccines for the prophylaxis of infectious diseases,on the other hand, for the preparation of heterologous proteins ineukaryotic cells.

Several such strains of vaccinia virus have been developed to avoidundesired side effects of smallpox vaccination. Thus, a modifiedvaccinia Ankara (MVA) has been generated by long-term serial passages ofthe Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts(for review see Mayr, A. et al. 1975 Infection 3:6-14; Swiss Patent No.568,392). The MVA virus is publicly available from American Type CultureCollection as ATCC No.: VR-1508. MVA is distinguished by its greatattenuation, as demonstrated by diminished virulence and reduced abilityto replicate in primate cells, while maintaining good immunogenicity.The MVA virus has been analyzed to determine alterations in the genomerelative to the parental CVA strain. Six major deletions of genomic DNA(deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs havebeen identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). Theresulting MVAvirus became severely host cell restricted to avian cells.

Furthermore, MVA is characterized by its extreme attenuation. Whentested in a variety of animal models, MVA was proven to be avirulenteven in immunosuppressed animals. More importantly, the excellentproperties of the MVA strain have been demonstrated in extensiveclinical trials (Mayr A. et al. 1978 Zentralbl Bakteriol [B]167:375-390; Stickl et al. 1974 Dtsch Med Wschr 99:2386-2392). Duringthese studies in over 120,000 humans, including high-risk patients, noside effects were associated with the use of MVA vaccine.

MVA replication in human cells was found to be blocked late in infectionpreventing the assembly to mature infectious virions. Nevertheless, MVAwas able to express viral and recombinant genes at high levels even innon-permissive cells and was proposed to serve as an efficient andexceptionally safe gene expression vector (Sutter, G. and Moss, B. 1992PNAS USA 89:10847-10851). Additionally, novel vaccinia vector vaccineswere established based on MVA having foreign DNA sequences inserted atthe site of deletion III within the MVA genome (Sutter, G. et al. 1994Vaccine 12:1032-1040).

Recombinant MVA vaccinia viruses can be prepared as set out hereinafter.A DNA-construct which contains a DNA-sequence which codes for a foreignpolypeptide flanked by MVA DNA sequences adjacent to a predeterminedinsertion site (e.g., between two conserved essential MVA genes such asI8R/G1L; in restructured and modified deletion III; or at othernon-essential sites within the MVA genome) is introduced into cellsinfected with MVA, to allow homologous recombination. Once theDNA-construct has been introduced into the eukaryotic cell and theforeign DNA has recombined with the viral DNA, it is possible to isolatethe desired recombinant vaccinia virus in a manner known per se,preferably with the aid of a marker. The DNA-construct to be insertedcan be linear or circular. A plasmid or polymerase chain reactionproduct is preferred. Such methods of making recombinant MVA vectors aredescribed in PCT publications WO/2006/026667 and WO/2016/115116incorporated by reference herein. The DNA-construct contains sequencesflanking the left and the right side of a naturally occurring deletion.The foreign DNA sequence is inserted between the sequences flanking thenaturally occurring deletion. For the expression of a DNA sequence orgene, it is necessary for regulatory sequences, which are required forthe transcription of the gene, to be present on the DNA. Such regulatorysequences (called promoters) are known to those skilled in the art, andinclude for example those of the vaccinia 11 kDa gene as are describedin EP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385). TheDNA-construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al. 1973 Virol 52:456-467; Wigler et al. 1979 Cell16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J.1:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol101:482-492), by means of liposomes (Straubinger et al. 1983 MethEnzymol 101:512-527), by means of spheroplasts (Schaffher 1980 PNAS USA77:2163-2167) or by other methods known to those skilled in the art.

The MVA vectors described and tested herein were unexpectedly found tobe effective after a single prime or a homologous prime/boost regimen.Other MVA vector designs require a heterologous prime/boost regimen,while still other published studies have been unable to induce effectiveimmune responses with MVA vectors. Conversely, the present MVA vectordesign and methods of manufacture are useful in producing effective MVAvaccine vectors for eliciting effective T-cell and antibody immuneresponses. Furthermore, the utility of an MVA vector capable ofeliciting effective immune responses and antibody production after asingle homologous prime boost is significant for considerations such asuse, commercialization and transport of materials especially to affectedthird world locations.

In one embodiment, the present invention is a recombinant viral vector(e.g., an MVA vector) comprising one or more heterologous gene insertsof a hepatitis B virus. The viral vector (e.g., an MVA vector) may beconstructed using conventional techniques known to one of skill in theart. The one or more heterologous gene inserts encode a polypeptidehaving desired immunogenicity, i.e., a polypeptide that can induce animmune reaction, cellular immunity and/or humeral immunity, in vivo byadministration thereof. The gene region of the vector (e.g., an MVAvector) where the gene encoding a polypeptide having immunogenicity isintroduced is flanked by regions that are indispensable. In theintroduction of a gene encoding a polypeptide having immunogenicity, anappropriate promoter may be operatively linked upstream of the geneencoding a polypeptide having desired immunogenicity.

The one or more genes may be selected from hepatitis B virus. In oneembodiment, the one more genes are selected from a hepatitis B virusgenotype. In exemplary embodiments, the gene encodes a polypeptide orprotein capable of inducing an immune response in the subject to whichit is administered, and more particularly, an immune response capable ofproviding a protective and/or therapeutic benefit to the subject. In oneembodiment, the one or more genes encode the virus premembrane proteinPreS2_S, PreS.HA or one or more nonstructural proteins PreCore/Core,truncated X, or M1.P41A. The heterologous gene inserts are inserted intoone or more deletion sites of the vector under the control of promoterscompatible with poxvirus expression systems or into a site between twoconserved essential MVA gene (e.g., 18R and G1L) of the vector under thecontrol of promoters compatible with poxvirus expression systems.

In one embodiment, the deletion Ill site is restructured and modified toremove non-essential flanking sequences.

In exemplary embodiments, the vaccine is constructed to express ahepatitis B virus PreS2_S protein (PrS2_S), which is inserted betweentwo conserved essential MVA genes (18R and G1L) using shuttle vectorpLW73-preS2_S; and to express hepatitis B virus PreCore/Core/TruncatedX, which is inserted into deletion Ill using shuttle vectorpLW76-preCore/Core-tr.X. These two shuttle vectors are constructed withan ampicillin resistance marker, allowing the vector to replicate inbacteria; with two flanking sequences, allowing the vector to recombinewith a specific location in the MVA genome; with a green fluorescentprotein (GFP) selection marker, allowing the selection of recombinantMVAs; with a sequence homologous to part of Flank 1 of the MVA sequence,enabling removal of the GFP sequence from the MVA vector after insertionof nonstructural gene into the MVA genome; with a modified HS (mH5)promoter, which enables transcription of the inserted heterologous geneinsert or with another promoter which enables transcription of theinserted heterologous gene insert; and with a hepatitis B gene.

In certain embodiments, the polypeptide, or the nucleic acid sequenceencoding the polypeptide, may have a mutation or deletion (e.g., aninternal deletion, truncation of the amino- or carboxy-terminus, or apoint mutation).

The one or more genes introduced into the recombinant viral vector areunder the control of regulatory sequences that direct its expression ina cell.

The nucleic acid material of the viral vector may be encapsulated, e.g.,in a lipid membrane or by structural proteins (e.g., capsid proteins),that may include one or more viral polypeptides.

In exemplary embodiments, the present invention is a recombinant viralvector (e.g., a recombinant MVA vector) comprising one or more genes, orone or more polypeptides encoded by the gene or genes, from a hepatitisB virus. The hepatitis B virus gene may encode a polypeptide or proteincapable of inducing an immune response in the subject to which it isadministered, and more particularly, an immune response capable ofproviding a protective and/or therapeutic benefit to the subject.

In certain embodiments, the one or more genes encodes a polypeptide, orfragment thereof, that is substantially identical (e.g., at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to theselected hepatitis B virus PreS2_S over at least 20, 25, 30, 35, 40, 45,50, 55, 60, 65, or 70 contiguous residues of the selected hepatitis Bvirus PreS2_S that retain immunogenic activity.

In certain embodiments, the one or more genes encodes a polypeptide, orfragment thereof, that is substantially identical (e.g., at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical) to theselected hepatitis B virus fusion protein M1.P41A over at least 20, 25,30, 35, 40, 45, 50, 55, 60, 65, or 70 contiguous residues of theselected hepatitis B virus fusion protein M1.P41A that retainsimmunogenic activity.

In one embodiment, the structural protein or fusion protein sequence isinserted into deletion site I, II, III, IV, V or VI of the MVA vector,and the nonstructural protein or fusion protein sequence is insertedinto deletion site I, II, III, IV, V or VI of the MVA vector.

In one embodiment, the structural protein or fusion protein sequence isinserted between I8R and Gil, of the MVA vector, or into restructuredand modified deletion Ill of the MVA vector; and the nonstructuralprotein or fusion protein sequence is inserted between I8R and Gil, ofthe MVA vector, or into restructured and modified deletion site Ill ofthe MVA vector.

In exemplary embodiments, the present invention is a recombinant MVAvector comprising at least one heterologous gene insert (e.g., one ormore gene inserts) from a hepatitis B virus which is under the controlof regulatory sequences that direct its expression in a cell. The genemay be, for example, under the control of a promoter selected from thegroup consisting of Pm2H5, Psyn II, or mH5 promoters.

One or more genes may be optimized for use in the MVA vector.Optimization includes codon optimization, which employs silent mutationsto change selected codons from the native sequences into synonymouscodons that are optimally expressed by the host-vector system. Othertypes of optimization include the use of silent mutations to interrupthomopolymer stretches or transcription terminator motifs. Each of theseoptimization strategies can improve the stability of the gene, improvethe stability of the transcript, or improve the level of proteinexpression from the gene. In exemplary embodiments, the number ofhomopolymer stretches in the sequence is reduced to stabilize theconstruct. A silent mutation may be provided for anything similar to avaccinia termination signal.

In exemplary embodiments, optimization of genes may include interruptinghomopolymer sequences (≥4 G/C and ≥A/T) by silent mutations, adding asecond TAA stop codon, or adding a Vaccinia Transcription TerminatorSequence at the end of the gene such as TTTTTAT.

In exemplary embodiments, the hepatitis structural or nonstructuralsequences are codon optimized for expression in MVA using a computeralgorithm; PrM-E and NS1 sequences with runs of ≥5 deoxyguanosines, ≥5deoxycytidines, ≥5 deoxyadenosines, and ≥5 deoxythymidines areinterrupted by silent mutation to minimize loss of expression due toframe shift mutations.

The recombinant viral vectors of the present invention may be used aloneor in combination. In one embodiment, two different recombinant viralvectors are used in combination, where the difference may refer to theone or more heterologous gene inserts or the other components of therecombinant viral vector or both. In exemplary embodiments, two or morerecombinant viral vectors are used in combination in order to protectagainst infection by hepatitis B in humans.

The present invention also extends to host cells comprising therecombinant viral vector described above, as well as isolated virionsprepared from host cells infected with the recombinant viral vector.

IV. Pharmaceutical Composition

The recombinant viral vectors of the present invention are readilyformulated as pharmaceutical compositions for veterinary or human use,either alone or in combination. The pharmaceutical composition maycomprise a pharmaceutically acceptable diluent, excipient, carrier, oradjuvant.

In one embodiment, the present invention is a vaccine effective toprotect and/or treat a hepatitis B virus infection comprising arecombinant MVA vector that expresses at least one hepatitis Bpolypeptide or an immunogenic fragment thereof. The vaccine compositionmay comprise one or more additional therapeutic agents.

The pharmaceutical composition may comprise 1, 2, 3, 4 or more than 4different recombinant MVA vectors.

In a particular embodiment, the first nucleic sequence encodes PreS2_Sor PreS.HA, and the first nucleic acid sequence of the first recombinantMVA vector is from the same or a different genotype than the firstnucleic acid sequence of the second recombinant MVA vector.

In one embodiment, the first and second sequences of the firstrecombinant MVA vector are from genotype Band the first and secondsequences of the second recombinant MVAvector are from genotype C.

In one embodiment, the first and second sequences of the firstrecombinant MVA vector are from genotype A and the first and secondsequences of the second recombinant MVA vector are from genotype D.

In one embodiment, the first and second sequences of the firstrecombinant MVA vector are from genotype C and the first and secondsequences of the second recombinant MVA vector are from genotype D.

In one embodiment, the pharmaceutical composition comprises fourrecombinant MVA vectors where the first and second sequences of each ofthe four vectors are from genotypes A, B, C, and D respectively.

As used herein, the phrase “pharmaceutically acceptable carrier”encompasses any suitable pharmaceutical carrier, such as those suitablefor parenteral administration, such as, for example, by intramuscular,intraarticular (in the joints), intravenous, intradermal,intraperitoneal, and subcutaneous routes. Examples of such formulationsinclude aqueous and non-aqueous, isotonic sterile injection solutions,which contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. One exemplary pharmaceutically acceptable carrier isphysiological saline.

Other physiologically acceptable diluents, excipients, carriers, oradjuvants and their formulations are known to those skilled in the art.

The compositions utilized in the methods described herein can beadministered by a route any suitable method, e.g., parenteral,intramuscular, intraarterial, intravascular, intravenous,intraperitoneal, subcutaneous, dermal, transdermal, ocular, inhalation,buccal, sublingual, perilingual, nasal, topical administration, and oraladministration. The preferred method of administration can varydepending on various factors (e.g., the components of the compositionbeing administered and the severity of the condition being treated).Formulations suitable for oral administration may consist of liquidsolutions, such as an effective amount of the composition dissolved in adiluent (e.g., water, saline, or PEG-400), capsules, sachets or tablets,each containing a predetermined amount of the vaccine. Thepharmaceutical composition may also be an aerosol formulation forinhalation, e.g., to the bronchial passageways. Aerosol formulations maybe mixed with pressurized, pharmaceutically acceptable propellants(e.g., dichlorodifluoromethane, propane, or nitrogen).

For the purposes of this invention, pharmaceutical compositions suitablefor delivering a therapeutic or biologically active agent can include,e.g., tablets, gelcaps, capsules, pills, powders, lyophilized powders,granulates, suspensions, emulsions, solutions, gels, hydrogels, oralgels, pastes, eye drops, ointments, creams, plasters, drenches, deliverydevices, microneedles, suppositories, enemas, injectables, implants,sprays, or aerosols. Any of these formulations can be prepared bywell-known and accepted methods of art. See, for example, Remington: TheScience and Practice of Pharmacy (21.sup.st ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2005, and Encyclopedia of PharmaceuticalTechnology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which ishereby incorporated by reference.

The immunogenicity of the composition (e.g., vaccine) may besignificantly improved if the composition of the present invention isco-administered with an immunostimulatory agent or adjuvant. Suitableadjuvants well-known to those skilled in the art include, e.g., aluminumphosphate, aluminum hydroxide, QS21, Quil A (and derivatives andcomponents thereof), calcium phosphate, calcium hydroxide, zinchydroxide, glycolipid analogs, octodecyl esters of an amino acid,muramyl dipeptides, polyphosphazene, lipoproteins, ISCOM-Matrix,DC-Chol, ODA, cytokines, and other adjuvants and derivatives thereof.

Pharmaceutical compositions according to the present invention may beformulated to release the composition immediately upon administration(e.g., targeted delivery) or at any predetermined time period afteradministration using controlled or extended release formulations.Administration of the pharmaceutical composition in controlled orextended release formulations is useful where the composition, eitheralone or in combination, has (i) a narrow therapeutic index (e.g., thedifference between the plasma concentration leading to harmful sideeffects or toxic reactions and the plasma concentration leading to atherapeutic effect is small; generally, the therapeutic index, Tl, isdefined as the ratio of median lethal dose (LD₅₀) to median effectivedose (ED₅₀); (ii) a narrow absorption window in the gastro-intestinaltract; or (iii) a short biological half-life, so that frequent dosingduring a day is required in order to sustain a therapeutic level.

Many strategies can be pursued to obtain controlled or extended releasein which the rate of release outweighs the rate of metabolism of thepharmaceutical composition. For example, controlled release can beobtained by the appropriate selection of formulation parameters andingredients, including, e.g., appropriate controlled releasecompositions and coatings. Suitable formulations are known to those ofskill in the art. Examples include single or multiple unit tablet orcapsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, lyophilization withencapsulation into solid dissolvable carriers, lyophilization withencapsulation into that substrates incorporated into microneedles,lyophilization with encapsulation into that substrates incorporated intomicroneedle patches, and liposomes.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the vaccine dissolved indiluents, such as water, saline or PEG 400; (b) capsules, sachets ortablets, each containing a predetermined amount of the vaccine, asliquids, solids, granules or gelatin; (c) suspensions in an appropriateliquid; (d) suitable emulsions; and (e) polysaccharide polymers such aschitins. The vaccine, alone or in combination with other suitablecomponents, may also be made into aerosol formulations to beadministered via inhalation, e.g., to the bronchi al passageways.Aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the vaccine with a suppository base.Suitable suppository bases include natural or synthetic triglycerides orparaffin hydrocarbons. In addition, it is also possible to use gelatinrectal capsules which consist of a combination of the vaccine with abase, including, for example, liquid triglycerides, polyethyleneglycols, and paraffin hydrocarbons.

Pharmaceutical compositions comprising any of the nucleic acid moleculesencoding hepatitis B viral proteins of the present invention are usefulto immunize a subject against disease caused by hepatitis B virusinfection. Thus, this invention further provides methods of immunizing asubject against disease caused by hepatitis B infection, comprisingadministering to the subject an immunoeffective amount of apharmaceutical composition of the invention. This subject may be ananimal, for example a mammal, such as a primate or preferably a human.

In various embodiments, the vaccines of the present invention may alsobe co-administered with cytokines to further enhance immunogenicity. Thecytokines may be administered by methods known to those skilled in theart, e.g., as a nucleic acid molecule in plasmid form or as a protein orfusion protein.

A. Immune Checkpoint Blockade

In various embodiments, the vaccines of the present invention may alsobe co-administered with checkpoint inhibitor agonists to further enhanceimmunogenicity.

The phenomenon of immune exhaustion was first identified in chroniclymphocytic choriomeningitis virus (LMCV) in mice and was later found tooccur in other human chronic viral infections such as HIV, HCV, and HBV,as well as in various cancers. A hallmark of T cell exhaustion in bothsuch viral infections and cancer is the increased expression of variousinhibitory receptors such as programmed death-1 (PD-1), cytotoxicT-lymphocyte antigen-4 (CTLA-4), cluster of differentiation 244 (CD244),cluster of differentiation 160 (CD160), and others. In cancerimmunotherapy, the use of checkpoint inhibitors such as those that blockthe PD-1:PD-L1 pathway has resulted in significant clinical benefitswith a wide range of cancer types including melanoma, non-small celllung cancer (NSCLC), and renal cell carcinoma (RCC). The fact that Tcell exhaustion is a major factor in allowing both the progression ofthese cancers and the persistence of chronic viral infections like HBVsuggests that checkpoint inhibitors may potentially achieve clinicalbenefits when used as treatments for chronic HBV.

In various embodiments, the compositions of the present invention mayalso be co-administered or sequentially administered with checkpointinhibitors.

Checkpoint inhibitors act by blocking a negative regulator of T-cellactivation and response and these inhibitors include any agent thatblocks or inhibits in a statistically significant manner, the inhibitorypathways of the immune system. Such inhibitors may include smallmolecule inhibitors or may include antibodies, or antigen bindingfragments thereof, that bind to and block or inhibit immune checkpointreceptors or antibodies that bind to and block or inhibit immunecheckpoint receptor ligands. Illustrative checkpoint molecules that maybe targeted for blocking or inhibition include, but are not limited to,CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3,VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressedon all NK, yo, and memory CD8⁺ (αβ) T cells), CD160 (also referred to asBY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 familyligands. B7 family ligands include, but are not limited to, B7-1, B7-2,B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpointinhibitors include antibodies, or antigen binding fragments thereof,other binding proteins, biologic therapeutics or small molecules, thatbind to and block or inhibit the activity of one or more of CTLA-4,PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160and CGEN-15049. Illustrative immune checkpoint inhibitors includeTremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-LI monoclonalAntibody (Anti-B7-HI; MEDI4736), MK-3475 (PD-1 blocker), Nivolumab(anti-PDI antibody), CT-011 (anti-PD1 antibody), BY55 monoclonalantibody, AMP224 (anti-PDLI antibody), BMS-936559 (anti-PDLI antibody),MPLDL3280A (anti-PDLI antibody), MSB0010718C (anti-PDLI antibody) andYervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint proteinligands include, but are not limited to PD-LI, PD-L2, B7-H3, B7-H4,CD28, CD86 and TIM-3.

In one specific embodiment, the vectors are administered in combinationwith, or sequentially with immune checkpoint blockade agent that blockthe interaction between immune checkpoint receptor programmed cell deathprotein 1 (PD-1) and its ligand PDL-1. See A. Mullard, “New checkpointinhibitors ride the immunotherapy tsunami,” Nature Reviews: DrugDiscovery (2013), 12:489-492. PD-1 is expressed on and regulates theactivity of T-cells. Specifically, when PO-1 is unbound to PDL-1, theT-cells can engage and kill target cells. However, when PD-1 is bound toPDL-1 it causes the T-cells to cease engaging and killing target cells.Furthermore, unlike other checkpoints, PD-1 acts proximately such thePDLs are overexpressed directly on cancer cells which leads to increasedbinding to the PD-1 expressing T-cells.

One aspect of the present disclosure provides checkpoint inhibitorswhich are antibodies that can act as agonists of PD-1, therebymodulating immune responses regulated by PD-1. In one embodiment, theanti-PD-1 antibodies can be antigen-binding fragments. Anti-PD-1antibodies disclosed herein are able to bind to human PD-1 and activatePD-1, thereby inhibiting the function of immune cells expressing PD-1.In one embodiment, the PD-1 agonist antibody selected from BMS 936558(nivolumab), BMS 936559, MK 3475, MPDL 3280A, AMP 224, or Medi 4736.

In one specific embodiment, the vectors are administered in combinationwith, or sequentially with immune checkpoint blockade agent that inhibitCTLA-4. Suitable anti-CTLA4 antagonist agents for use in the methods ofthe invention, include, without limitation, anti-CTLA4 antibodies, humananti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4antibodies, humanized anti-CTLA4 antibodies, monoclonal anti-CTLA4antibodies, polyclonal anti-CTLA4 antibodies, chimeric anti-CTLA4antibodies, MDX-010 (ipilimumab), tremelimumab, anti-CD28 antibodies,anti-CTLA4 adnectins, anti-CTLA4 domain antibodies, single chainanti-CTLA4 fragments, heavy chain anti-CTLA4 fragments, light chainanti-CTLA4 fragments, inhibitors of CTLA4 that agonize theco-stimulatory pathway, the antibodies disclosed in PCT Publication No.WO 2001/014424, the antibodies disclosed in PCT Publication No. WO2004/035607, the antibodies disclosed in U.S. Publication No.2005/0201994, and the antibodies disclosed in granted European PatentNo. EP 1212422 81. Additional CTLA-4 antibodies are described in U.S.Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCTPublication Nos. WO 01/14424 and WO 00/37504; and in U.S. PublicationNos. 2002/0039581 and 2002/086014. Other anti-CTLA-4 antibodies that canbe used in a method of the present invention include, for example, thosedisclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and 6,207,156;Hurwitz et al, Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998);Camacho et al, J. Clin. Oncology, 22(145):Abstract No. 2505 (2004)(antibody CP-675206); Mokyr et al, Cancer Res., 58:5301-5304 (1998), andU.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.

Additional anti-CTLA4 antagonists include, but are not limited to, thefollowing: any inhibitor that is capable of disrupting the ability ofCD28 antigen to bind to its cognate ligand, to inhibit the ability ofCTLA4 to bind to its cognate ligand, to augment T cell responses via theco-stimulatory pathway, to disrupt the ability of B7 to bind to CD28and/or CTLA4, to disrupt the ability of B7 to activate theco-stimulatory pathway, to disrupt the ability of CD80 to bind to CD28and/or CTLA4, to disrupt the ability of CD80 to activate theco-stimulatory pathway, to disrupt the ability of CD86 to bind to CD28and/or CTLA4, to disrupt the ability of CD86 to activate theco-stimulatory pathway, and to disrupt the co-stimulatory pathway, ingeneral from being activated. This necessarily includes small moleculeinhibitors of CD28, CD80, CD86, CTLA4, among other members of theco-stimulatory pathway; antibodies directed to CD28, CD80, CD86, CTLA4,among other members of the co-stimulatory pathway; antisense moleculesdirected against CD28, CD80, CD86, CTLA4, among other members of theco-stimulatory pathway; adnectins directed against CD28, CD80, CD86,CTLA4, among other members of the co-stimulatory pathway, RNAiinhibitors (both single and double stranded) of CD28, CD80, CD86, CTLA4,among other members of the co-stimulatory pathway, among otheranti-CTLA4 antagonists.

In one specific embodiment, the vectors are administered in combinationwith, or sequentially with immune checkpoint blockade agent that inhibitTIM-3. Blocking the activation of TIM-3 by a ligand, results in anincrease in ThI cell activation. Furthermore, TIM-3 has been identifiedas an important inhibitory receptor expressed by exhausted CD8⁺ T cells.TIM-3 has also been reported as a key regulator of nucleic acid mediatedantitumor immunity. In one example, TIM-3 has been shown to beupregulated on tumor-associated dendritic cells (TADCs).

This invention also provides kits comprising the vaccines of the presentinvention. For example, kits comprising a vaccine and instructions foruse are within the scope of this invention.

V. Method of Use

The compositions of the invention can be used as vaccines for inducingan immune response to a hepatitis B virus.

In exemplary embodiments, the present invention provides a method ofpreventing a hepatitis B infection to a subject in need thereof (e.g.,an unexposed subject), comprising administering the composition of thepresent invention to the subject in a prophylactically effective amount.The result of the method is that the subject is partially or completelyimmunized against the virus.

In exemplary embodiments, the present invention provides a method oftreating a hepatitis B infection in a subject in need thereof (e.g., anexposed subject, such as a subject who has been recently exposed but isnot yet symptomatic, or a subject who has been recently exposed and isonly mildly symptomatic, or a subject who has been recently exposed andis strongly symptomatic, or a subject who was long ago exposed and isweakly or strongly symptomatic), comprising administering thecomposition of the present invention to the subject in a therapeuticallyeffective amount. The result of treatment is a subject that has animproved therapeutic profile.

In exemplary embodiments, the present invention provides a method oftreating a hepatitis B infection in a subject in need thereof (e.g., anexposed subject who is in the chronic stages of infection), comprisingadministering the composition of the present invention to the subject ina therapeutically effective amount. The result of treatment is a subjectthat has an improved therapeutic profile.

Typically, the vaccines will be in an admixture and administeredsimultaneously, but may also be administered separately.

A subject to be treated according to the methods described herein (e.g.,a subject infected with, a hepatitis B virus) may be one who has beendiagnosed by a medical practitioner as having such a condition.Diagnosis may be performed by any suitable means. A subject in whom thedevelopment of an infection is being prevented may or may not havereceived such a diagnosis. One skilled in the art will understand that asubject to be treated according to the present invention may have beenidentified using standard tests or may have been identified, withoutexamination, as one at high risk due to the presence of one or more riskfactors (e.g., exposure to hepatitis B virus, etc.).

Prophylactic treatment may be administered, for example, to a subjectnot yet exposed to or infected by a hepatitis B virus but who issusceptible to, or otherwise at risk of exposure or infection with anhepatitis B virus.

Therapeutic treatment may be administered, for example, to a subjectalready exposed to or infected by a hepatitis B who is not yet ill, orshowing symptoms or infection, suffering from a disorder in order toimprove or stabilize the subject's condition (e.g., a patient alreadyinfected with a hepatitis B virus). The result is an improvedtherapeutic profile. In some instances, as compared with an equivalentuntreated control, treatment may ameliorate a disorder or a symptomthereof by, e.g., about 5%, about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, orabout 100% as measured by any standard technique. In some instances,treating can result in the inhibition of viral replication, a decreasein viral titers or viral load, eradication or clearing of the virus.Therapeutic treatment may be administered, for example, to a subjectalready exposed to or infected by a hepatitis B who is in the chronicstages of infection (e.g., a patient already infected with a hepatitis Bvirus). The result is an improved therapeutic profile. In someinstances, as compared with an equivalent untreated control, treatmentmay ameliorate a disorder or a symptom thereof by, e.g., about 5%, about10%, about 20%, about 30%, about about 40%, about 50%, about 60%, about70%, about 80%, about 90%, about 95%, or about 100% as measured by anystandard technique. In some instances, treating can result in theinhibition of viral replication, a decrease in viral titers or viralload, eradication or clearing of the virus.

In other embodiments, treatment may result in amelioration of one ormore symptoms of the infection, including any symptom identified above.According to this embodiment, confirmation of treatment can be assessedby detecting an improvement in or the absence of symptoms.

In other embodiments, treatment may result in reduction or eliminationof the ability of the subject to transmit the infection to another,uninfected subject. Confirmation of treatment according to thisembodiment is generally assessed using the same methods used todetermine amelioration of the disorder, but the reduction in viral titeror viral load necessary to prevent transmission may differ from thereduction in viral titer or viral load necessary to ameliorate thedisorder.

In one embodiment, the present invention is a method of inducing animmune response in a subject (e.g., a human) by administering to thesubject a recombinant viral vector that encodes at least one gene from ahepatitis B virus. The immune response may be a cellular immuneresponse, a humeral immune response or a combination thereof. The immuneresponse may be a T-cell response, a B-cell response or an antibodyresponse or a combination thereof.

In a particular embodiment, the present invention is a method ofinducing an immune response in a subject (e.g., a human) in need thereofby administering to the subject a recombinant viral vector that encodesat least one gene from a hepatitis B virus. The composition may beadministered, e.g., by injection (e.g., intramuscular, intraarterial,intravascular, intravenous, intraperitoneal, or subcutaneous).

It will be appreciated that more than one route of administering thevaccines of the present invention may be employed either simultaneouslyor sequentially (e.g., boosting). In addition, the vaccines of thepresent invention may be employed in combination with traditionalimmunization approaches such as employing protein antigens, vacciniavirus and inactivated virus, as vaccines. Thus, in one embodiment, thevaccines of the present invention are administered to a subject (thesubject is “primed” with a vaccine of the present invention) and then atraditional vaccine is administered (the subject is “boosted” with atraditional vaccine). In another embodiment, a traditional vaccine isfirst administered to the subject followed by administration of avaccine of the present invention. In yet another embodiment, atraditional vaccine and a vaccine of the present invention areco-administered.

It will also be appreciated that single or multiple administrations ofthe vaccine compositions of the present invention may be carried out.For example, subjects who are particularly susceptible to hepatitis Bvirus infection may require multiple immunizations to establish and/ormaintain protective immune responses. Levels of induced immunity can bemonitored by measuring amounts of binding and neutralizing secretory andserum antibodies as well as levels of T cells, and dosages adjusted orvaccinations repeated as necessary to maintain desired levels ofprotection.

In one embodiment, administration is repeated at least once, at leasttwice, at least 3 times, at least 4 times, at least 5 times, at least 6times, at least 7 times, at least 8 times, or more than 8 times.

In one embodiment, administration is repeated once.

In one embodiment, administration is repeated twice.

In one embodiment, about 2-8, about 4-8, or about 6-8 administrationsare provided.

In one embodiment, about 1-4-week, 2-4 week, 3-4 week, 1 week, 2 week, 3week, 4 week or more than 4 week intervals are provided betweenadministrations.

In one specific embodiment, a 4-week interval is used between 2administrations.

In one specific embodiment, a 4-week interval is used between eachadministration of 3 total administrations.

In an exemplary treatment strategy, the invention provides a method oftreating HBV infection in a subject in need thereof by:

-   -   1) administering an effective amount of an antiretroviral or        nucleoside analog composition to reduce viral loads;    -   2) administering an immunogenic composition to prime an immune        response to HBV; and    -   3) administering an immunogenic composition to boost an immune        response to HBV to treat one or more symptoms of HBV infection.

In one embodiment, an immune checkpoint inhibitor is administered beforethe immunogenic composition.

In one embodiment, an immune checkpoint inhibitor is administeredconcurrently with the immunogenic composition.

In one embodiment, an immune checkpoint inhibitor is administered afterthe immunogenic composition.

In one embodiment, the immunogenic compositions induces anti-HBV T celland/or B cell responses.

In one embodiment, the method rescues exhausted T cells and maintains Tcell function.

In various embodiments, the methods are continued to obtain selectendpoints that are indicative of efficacy of the immunogeniccompositions described herein.

In one embodiment, the immunogenic composition is administered to changethe HBsAg status of a subject infected with HBV from positive tonegative.

In one embodiment, the immunogenic composition is administered to changethe status of detectable levels of circulating HBsAg of a subjectinfected with HBV from positive to negative.

In another embodiment, the immunogenic composition is administered toinduce the formation of neutralizing antibodies and antibody-dependentcell-mediated cytotoxicity (ADCC).

In another embodiment, the immunogenic composition is administered toinduce CD4⁺ helper and CD8⁺ CTL responses.

In another embodiment, the immunogenic composition is administered toreduce or eliminate viral load to undetectable levels and prevent orreduce inflammation in a subject.

A. Dosage

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount, as will be therapeutically effective,immunogenic and protective. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theimmune system of the individual to synthesize antibodies, and, ifneeded, to produce a cell-mediated immune response. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and may be monitored on a patient-by-patient basis.However, suitable dosage ranges are readily determinable by one skilledin the art and generally range from about 5.0×10⁶ TCID₅₀ to about5.0×10⁹ TCID₅₀. The dosage may also depend, without limitation, on theroute of administration, the patient's state of health and weight, andthe nature of the formulation.

The pharmaceutical compositions of the invention are administered insuch an amount as will be therapeutically effective, immunogenic, and/orprotective against a pathogenic species of hepatitis B virus. The dosageadministered depends on the subject to be treated (e.g., the manner ofadministration and the age, body weight, capacity of the immune system,and general health of the subject being treated). The composition isadministered in an amount to provide a sufficient level of expressionthat elicits an immune response without undue adverse physiologicaleffects. Preferably, the composition of the invention is a heterologousviral vector that includes one or more polypeptides of the hepatitis Bvirus, or a nucleic acid molecule encoding one or more genes of thehepatitis B virus, and is administered at a dosage of, e.g., between1.0×10⁴ and 9.9×10¹² TCID₅₀ of the viral vector, preferably between1.0×10⁵ TCID₅₀ and 1.0×10¹¹ TCID₅₀, more preferably between 1.0×10⁶ and1.0×10¹⁰ TCID₅₀, or most preferably between 5.0×10⁶ and 5.0×10⁹ TCID₅₀.The composition may include, e.g., at least 5.0×10⁶ TCID₅₀ of the viralvector (e.g., 1.0×10⁸ TCID₅₀ of the viral vector). A physician orresearcher can decide the appropriate amount and dosage regimen. Thecomposition of the method may include, e.g., between 1.0×10⁴ and9.9×10¹² TCID₅₀ of the viral vector, preferably between 1.0×10⁵ TCID₅₀and 1.0×10¹¹ TCID₅₀, more preferably between 1.0×10⁶ and 1.0×10¹⁰TCID₅₀, or most preferably between 5.0×10⁶ and 5.0×10⁹ TCID₅₀. Thecomposition may include, e.g., at least 5.0×10⁶ TCID₅₀ of the viralvector (e.g., 1.0×10⁸ TCID₅₀ of the viral vector). The method mayinclude, e.g., administering the composition to the subject two or moretimes.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. However, a suitable dosagerange may be, for example, of the order of several hundred microgramsactive ingredient per vaccination. In other non-limiting examples, adose may also comprise from about 1 microgram/kg/body weight, about 5microgram/kg/body weight, about 10 microgram/kg/body weight, about 50microgram/kg/body weight, about 100 microgram/kg/body weight, about 200microgram/kg/body weight, about 350 microgram/kg/body weight, about 500microgram/kg/body weight, about 1 milligram/kg/body weight, about 5milligram/kg/body weight, about 10 milligram/kg/body weight, about 50milligram/kg/body weight, about 100 milligram/kg/body weight, about 200milligram/kg/body weight, about 350 milligram/kg/body weight, about 500milligram/kg/body weight, to about 1000 mg/kg/body weight or more pervaccination, and any range derivable therein. In non-limiting examplesof a derivable range from the numbers listed herein, a range of about 5mg/kg/body weight to about 100 mg/kg/body weight, about 5microgram/kg/body weight to about 500 milligram/kg/body weight, etc.,can be administered, based on the numbers described above. A suitableregime for initial administration and booster administrations (e.g.,inoculations) are also variable, but are typified by an initialadministration followed by subsequent inoculation(s) or otheradministration(s).

The invention also features a method of inducing an immune response tohepatitis B virus in a subject (e.g., a human) that includesadministering to the subject an effective amount of a recombinant viralvector that encodes at least one gene from hepatitis B virus. Thesubject being treated may not have, but rather be at risk of developing,an infection by a hepatitis B virus. Alternatively, the subject mayalready be infected with a hepatitis B virus. The composition may beadministered, e.g., by injection (e.g., intramuscular, intraarterial,intravascular, intravenous, intraperitoneal, or subcutaneous).

The term “effective amount” is meant the amount of a compositionadministered to improve, inhibit, or ameliorate a condition of asubject, or a symptom of a disorder, in a clinically relevant manner(e.g., improve, inhibit, or ameliorate infection by hepatitis B virus orprovide an effective immune response to infection by hepatitis B virus).Any improvement in the subject is considered sufficient to achievetreatment. Preferably, an amount sufficient to treat is an amount thatprevents the occurrence or one or more symptoms of hepatitis B virusinfection or is an amount that reduces the severity of, or the length oftime during which a subject suffers from, one or more symptoms ofhepatitis B virus infection (e.g., by at least 10%, 20%, or 30%, morepreferably by at least 50%, 60%, or 70%, and most preferably by at least80%, 90%, 95%, 99%, or more, relative to a control subject that is nottreated with a composition of the invention). A sufficient amount of thepharmaceutical composition used to practice the methods described herein(e.g., the treatment of hepatitis B virus infection) varies dependingupon the manner of administration and the age, body weight, and generalhealth of the subject being treated.

Ultimately, the prescribers or researchers will decide the appropriateamount and dosage. It is important to note that the value of the presentinvention may never be demonstrated in terms of actual clinical benefit.Instead, it is likely that the value of the invention will bedemonstrated in terms of success against a surrogate marker forprotection. For an indication such as hepatitis B virus infection, inwhich it is impractical or unethical to attempt to measure clinicalbenefit of an intervention, the FDA's Accelerated Approval processallows approval of a new vaccine based on efficacy against a surrogateendpoint. Therefore, the value of the invention may lie in its abilityto induce an immune response that constitutes a surrogate marker forprotection.

Similarly, FDA may allow approval of vaccines against hepatitis B virusbased on its Animal Rule. In this case, approval is achieved based onefficacy in animals. The value of the invention may lie in its abilityto protect relevant animal species against infection with hepatitis Bvirus, thus providing adequate evidence to justify its approval.

The composition of the method may include, e.g., between 1.0×10⁴ and9.9×10¹² TCID₅₀ of the viral vector, preferably between 1.0×10⁵ TCID₅₀and 1.0×10¹¹ TCID₅₀, more preferably between 1.0×10⁶ and 1.0×10¹⁰TCID₅₀, or most preferably between 5.0×10⁶ and 5.0×10⁹ TCID₅₀. Thecomposition may include, e.g., at least 5.0×10⁶ TCID₅₀ of the viralvector. The method may include, e.g., administering the composition twoor more times. In some instances, it may be desirable to combine thehepatitis B virus vaccines of the present invention with vaccines, whichinduce protective responses to other agents, particularly other viruses.For example, the vaccine compositions of the present invention can beadministered simultaneously, separately or sequentially with othergenetic immunization vaccines such as those for influenza (Ulmer, J. B.et al., Science 259:1745-1749 (1993); Raz, E. et al., PNAS (USA)91:9519-9523 (1994)), malaria (Doolan, D. L. et al., J. Exp. Med.183:1739-1746 (1996); Sedegah, M. et al., PNAS (USA) 91:9866-9870(1994)), and tuberculosis (Tascon, R. C. et al., Nat. Med. 2:888-892(1996)).

B. Administration Routes

As used herein, the term “administering” refers to a method of giving adosage of a pharmaceutical composition of the invention to a subject.The compositions utilized in the methods described herein can beadministered by a route selected from, e.g., parenteral, dermal,transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal,rectal, topical administration, and oral administration. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intraarterial, intravascular, and intramuscular administration. Thepreferred method of administration can vary depending on various factors(e.g., the components of the composition being administered and theseverity of the condition being treated).

Administration of the pharmaceutical compositions (e.g., vaccines) ofthe present invention can be by any of the routes known to one of skillin the art. Administration may be by, e.g., intramuscular injection. Thecompositions utilized in the methods described herein can also beadministered by a route selected from, e.g., parenteral, dermal,transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal,rectal, topical administration, and oral administration. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous, andintramuscular administration. The preferred method of administration canvary depending on various factors, e.g., the components of thecomposition being administered and the severity of the condition beingtreated.

In addition, single or multiple administrations of the compositions ofthe present invention may be given to a subject. For example, subjectswho are particularly susceptible to hepatitis B virus infection mayrequire multiple administrations to establish and/or maintain protectionagainst the virus. Levels of induced immunity provided by thepharmaceutical compositions described herein can be monitored by, e.g.,measuring amounts of neutralizing secretory and serum antibodies. Thedosages may then be adjusted or repeated as necessary to maintaindesired levels of protection against viral infection.

The claimed invention is further described by way of the followingnon-limiting examples. Further aspects and embodiments of the presentinvention will be apparent to those of ordinary skill in the art, inview of the above disclosure and following experimental exemplification,included by way of illustration and not limitation, and with referenceto the attached figures.

EXAMPLES Example 1: Construction of a Virus-Like Particle Vaccines forHepatitis B Virus Antigens

This example provides information on exemplary MVA vaccine vectors.Table 1 lists two exemplary MVA vaccine vectors.

TABLE 1 MVA vaccine vectors Vaccine Structural designation sequenceNon-Structural sequence GEO-HBV01 PreS2_S PreCore/Core + Truncated XGEO-HBV02 PreS.HA M1.P41A

For GEO0HBV01, the preS2_S sequence was cloned into pLW-73 MVA shuttlevector, placed under the control of vaccinia virus mH5 promoter. ThepLW73-preS2_S shuttle vector is used to insert the HBV preS2_S sequencesbetween essential genes I8R and G1L. The PreCore/Core-tr.X sequenceswere cloned into IPW-76 MVA shuttle vector. PreCore/Core is placed underthe control of vacinia virus mH5 promoter and the truncated X (tr.X)gene is under the control of vaccinia virus P7.5 promoter. The pLW76PreCore/Core-tr.X shuttle vector has been used to insert the HBVpreCore/Core and tr.X sequences into the modified deletion III site ofMVA (between the AS0R and B1R genes).

For GEO-HBV02, the preS.HA sequence was cloned into pLW-73 MVA shuttlevector, placed under the control of vaccinia virus mH5 promoter. ThepLW73-preS.HA shuttle vector is used to insert the HBV preS.HA sequencesbetween essential genes I8R and G1L. The M1.P41A sequences were clonedinto IPW-76 MVA shuttle vector. M1.P41A is placed under the control ofvacinia virus mH5 promoter. The pLW76 M1.P41A shuttle vector has beenused to insert the HBV M1.P41A sequence into the modified deletion Illsite of MVA (between the A50R and B1R genes).

Example 2: Evaluation of Virus-Like Particle Vaccines of the Hepatitis BVirus Antigens for Protection Protects Mice Against Challenge

The immunogenicity of HBV antigen VLP is assessed as a potential vaccinecandidate. Immunization with VLP MVA vaccine is evaluated for inductionboth potent humeral and cellular immune responses, and protection fromHBV challenge.

Materials and Methods Plasmids and Cells

The vectors for expressing HBV preS2_S or PreS.HA are described herein.293T cells are maintained in DMEM supplemented with 10% fetal bovineserum (FBS).

Indirect Immunofluorescence

293T cells are grown on glass coverslips and transfected with pGEO-HBV01and pGEO-HBV02 and 48 hr posttransfection, cells are fixed with 4%paraformaldehyde.

Cells are classified into two groups. One was permeabilized with 0.2%Triton X-100 for 5 min, the other without permeabilization. Afterblocking for 1 h in PBS containing 5% goat serum, all cells areincubated with poly clonal rabbit anti-preS sera at 4° C. overnight.Cells are washed with PBS following incubation with Alexa Fluor®488-Conjugated goat anti-rabbit secondary antibody for 1 h at 37° C.After washing, cells are stained with DAPI for 10 min, and then mountedonto microscope slides. Confocal slices are acquired with a 100×objective, using a Zeiss 510 confocal microscope with random sampling.

Preparation and Characterization of the Virus-Like Particles

The pGEO-HBV01 and pGEO-HBV02 plasmids are transfected into 293T cellswith polyethylenimine. 72 hr after transfection, the culture medium iscentrifuged at 6000 rpm for 15 min at 4° C. to remove cellular debris,followed by centrifugation at 22,000 rpm for 3 hr at 4° C. The pellet isresuspended in PBS at 4° C. overnight, and further purified through a20%-60% sucrose gradient in a Beckman SW41 Ti rotor at 30,000 rpm for 3hr at 4° C. The 40% sucrose fraction is harvested and diluted with PBSby about 5 fold. After centrifugation at 22,000 rpm for 3 hr at 4° C. toremove the sucrose, the virus-like particles are resuspended in PBS at4° C. overnight. A sample is applied to a 400 mesh carbon-coated coppergrid, and stained with 1% phosphotungstic acid (J&K Scientific). HBVantigenVLP are visualized on a Tecnai cJ2 Spirit transmission electionmicroscope operating at 120 kV.

LC-MSIMS Analysis

The expression of antigens is analyzed by LC-MS/MS. Briefly, 40% sucrosefraction are subjected to electrophoresis on a 12%-SDS-PAGE gel, whichis stained by coomassie R250. The coomassie R250 stained gel bands arecut, followed by in-gel digestion with trypsin [promega,enzyme:protein=1:50 (wt/wt)] at 37° C. for 12 h in 25 mM ammoniumbicarbonate buffer. The lyophilized tryptic digested samples arere-dissolved in 2% acetonitrile, 0.1% formic acid, and loaded on ChromXPC18 (3 μm, 120 A) nanoLC trap column. The online trapping, desaltingprocedure is carried out at a flow rate of 2 !iL/min for 10 min with100% solvent A (Solvent A: water/acetonitrile/formic acid=9812/0.1%solvent B: 2/98/0.1%). Then, an 60-min gradient elution ranging from5-35% acetonitrile (0.1% formic acid) is used on an analytical column(75 μm×15 cm C18-3 μm 120 A, ChromXP Eksigent). LC-MS/MS analysis isperformed with a Triple TOF 5600 System (AB SCIEX, Concord, ON) fittedwith a Nanospray III source (AB SCIEX, Concord, ON). Data is acquiredusing an ion spray voltage of 2.5 kV, curtain gas of 30 PSI, nebulizergas of 5 PSI, and an interface heater temperature of 150° C. The MS isoperated with TOF-MS scans. For IDA, survey scans were acquired in 250ms and as many as 25 product ion scans (90 ms) are collected ifexceeding a threshold of 150 counts per second (counts/s) and with a +2to +4 charge-state. A Rolling collision energy setting is applied to allprecursor ions for collision-induced dissociation. Dynamic exclusion wasset for Yz of peak width (˜12 s). For data analysis, the .wiff files areprocessed by ProteinPilot 5.0. Searches are performed against the localdatabase including the protein sequences for the HBV antigens using thedefault settings.

Immunization and Challenge

Female Balb/c mice of 6-8 weeks old are immunized by injecting the MVAvector in the hindlimb. A booster is given on day 22. Blood wascollected on day 52, and 112, and neutralizing antibody titers weredetermined by ELISA. On day 52, activated T cells in splenocytes orintrahepatic leukocytes are analyzed by ELISPOT and FACS. The immunizedmice are challenged on day 70. 10 !lg of pT-HBV1.3 (a plasmid containing1.3 genome length of HBV) is in hydrodynamic injection to establish HBVinfection as previously described (Yang P L, et al. Proc Nat! Acad SciUSA 2002 99: 13825-13830). Blood samples are collected at different timepoints to measure HBV antigens. On day 67, mice are sacrificed and livertissues are used for measuring antigens and RNA of HBV. Activated Tcells are also analyzed by FACS and ELISPOT assay. All mouse experimentsare conducted in accordance with the institutional guidelines followingthe experimental protocol reviewed and approved by university animalcontrol authority.

Isolation of Splenocytes and Intrahepatic Leukocytes

For the isolation of splenocytes, splenocytes are gently grindedfollowed by passaging through 40 um strainers and treating with ACKlysing buffer. After washing with PBS, cells were resuspended in DMEMsupplemented with 10% fetal bovine serum (FBS) and 1%Penicillin-Streptomycin-L-Glutamine. For the isolation of intrahepaticleukocytes, mice livers are perfused with pre-warmed Hanks' balancedsolution without Ca2+, Mg2+, followed by perfusing with 20 ml 0.025%collagenase D in Hanks' balanced salt solution, and let sit for 10 minat 37° C. Livers are then gently grinded followed by passaging through40 !-lm strainers. After centrifugation, cells are resuspended in 40%(vol/vol) Percell in DMEM, and layered over 70% Percell in PBS(vol/vol). After centrifugation of the gradient for 20 min at 2000 rpm,the cells at the interphase are collected. The cells are then treatedwith ACK lysing buffer, washed with PBS, and resuspended in DMEMsupplemented with 10% fetal bovine serum (FBS) and 1%Penicillin-Streptomycin-L-Glutamine for further analysis.

Enzyme-Linked Immunospot Assay

T cell responses are determined using an IFN-γ ELISPOT set (BOBiosciences) following the manufacturer's protocol. Briefly, 96-wellplates are coated with purified anti-mouse IFN−/, antibody (1:200) at 4°C. overnight, and then are blocked for 2 h using DMEM supplemented with10% fetal bovine serum (FBS) and 1% PenicillinStreptomycin-L-Glutamine.Splenocytes or intrahepatic leukocytes are seeded at 2×IO5/well.Peptides representing previously described epitopes present in proteinor purified protein are used to stimulate cells for 36 h at 37° C. in a5% CO2 and humidified incubator, with media and phorbol myristateacetate (PMA)/ionomycin-30 treated cells used as negative and positivecontrols, respectively. After being washed, cells are incubated withbiotinylated anti-mouse IFN-γ antibody (1:250) for 2 hat roomtemperature, and then incubated with streptavidin-horseradish peroxidase(HRP) (1:1,000) for 1 h. Following the final washes,3-amino-9-ethylcarbazole (AEC) substrate (Alfa Aesar) is added to thewells and allowed to develop at room temperature for 40 min. Thereaction is stopped with distilled water, and the plates are allowed toair dry 5 before spot-forming cells are enumerated by using an ELISPOTplate reader.

Flow Cytometry

Splenocytes or intrahepatic leukocytes are resuspended in DMEMsupplemented with 10% fetal bovine serum and 1%Penicillin-Streptomycin-L-Glutamine, and then are seeded at 2×10⁶/well.The cells are then stimulated for 6 h with preS-specific peptides orpurified recombinant preS diluted to a final concentration of 10 llg/mlin DMEM supplemented with 211 g/ml brefeldin A (BD Biosciences). Thecells were then washed in staining buffer (pBS containing 2% fetalbovine serum) and stained for CD8 and CD4 surface expression for 30 minat 4° C. using fluorescein isothiocyanate (FITC) conjugated anti-mouseCD8 antibody (BD Biosciences) and peridinin chlorophyll protein(perCP)-conjugated anti-mouse CD4 antibody (BD Biosciences). Then thecells are washed, fixed, and permeabilized using a commerciallyavailable Cytofix/Cytoperm kit (BD Biosciences). The cells are thenstained for 40 min at 4° C. for intracellular cytokine expression usingphycoerythrin (PE)-conjugated anti-mouse IFN-r antibody (BDBiosciences). After washing, cells are resuspended in staining bufferand analyzed using a BD FACS Canto™ II flow cytometer (BD Biosciences)and FACSDiva Version.

ELISA

Purified antigen (5 flg/ml) or preS VLP (1 flg/ml) is absorbed to 96well plates, blocked with 10% BSA, and then 50 fll of 1:100 dilution ofsera is incubated for 30 min at 37° C. followed by incubation with addedHRP-conjugated anti-mouse lgG, 5 lgG1 or lgG2a (Santa CruzBiotechnology) for 30 min at 37° C., and then with TMB substrate for 10minutes before stopping with 2 M H2S04 for measurement of opticaldensity at 450 nm. In addition, serum samples were diluted 1:5 for HBsAgang HBeAg detection.

Immunohistochemistry

Liver tissue is collected and fixed in 10% neutral formalin. Afterparaffin embedding, liver sections are used to detect HBV core antigenby immunohistochemical staining using polyclonal rabbit anti-HBcAgantibody (Dako).

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingdrawings and claims, that various changes, modifications and variationscan be made therein without departing from the spirit and scope of theinvention as defined in the following claims.

All references cited herein are incorporated by reference in theirentirety.

We claim:
 1. A modified vaccinia Ankara (MVA) vector comprising a firstnucleic acid sequence and a second nucleic acid sequence, wherein: thefirst nucleic acid sequence encodes a hepatitis B virus structuralprotein comprising a PreS2-S protein, wherein the first nucleic acidsequence is inserted into the MVA vector under the control of at leastone promoter compatible with a poxvirus expression system; the secondnucleic acid sequence hepatitis B non-structural proteins PreCore/Coreand Truncated X, and wherein the second nucleic acid sequence isinserted into the MVA vector under the control of at least one promotercompatible with a poxvirus expression system; and, wherein the hepatitisB virus structural protein and hepatitis B non-structural proteinsassemble into virus-like particles (VLPs) when expressed.
 2. Themodified vaccinia Ankara (MVA) vector of claim 1, wherein the firstnucleic acid sequence is inserted between MVA genes I8R and G1L.
 3. Themodified vaccinia Ankara (MVA) vector of claim 1, wherein the firstnucleic acid sequence is inserted between MVA genes I8R and G1L, and thesecond nucleic acid sequence is inserted in modified deletion III. 4.The modified vaccinia Ankara (MVA) vector of claim 1, wherein thepromotor is selected from the group consisting of Pm2H5, Psyn II, mH5,P7.5 promoters, or combinations thereof.
 5. The modified vaccinia Ankara(MVA) vector of claim 1, wherein the first nucleic acid sequencecomprises SEQ ID NO:
 1. 6. The modified vaccinia Ankara (MVA) vector ofclaim 1, wherein the second nucleic acid sequence comprises SEQ ID NO: 2and SEQ ID NO:
 3. 7. The modified vaccinia Ankara (MVA) vector of claim1, wherein the first nucleic acid sequence comprises SEQ ID NO: 1 andthe second nucleic acid sequence comprises SEQ ID NO: 2 and SEQ ID NO:3.
 8. A pharmaceutical composition comprising the modified vacciniaAnkara (MVA) vector of claim 1, and a pharmaceutically acceptablecarrier.
 9. A method for generating an immune response in a human tohepatitis B virus comprising administering to the human an effectiveamount of a pharmaceutical composition comprising a modified vacciniaAnkara (MVA) vector and a pharmaceutically acceptable carrier, whereinthe MVA vector comprising a first nucleic acid sequence and a secondnucleic acid sequence, wherein: the first nucleic acid sequence encodesa hepatitis B virus structural protein comprising a PreS2-S protein,wherein the first nucleic acid sequence is inserted into the MVA vectorunder the control of at least one promoter compatible with a poxvirusexpression system; the second nucleic acid sequence hepatitis Bnon-structural proteins PreCore/Core and Truncated X, and wherein thesecond nucleic acid sequence is inserted into the MVA vector under thecontrol of at least one promoter compatible with a poxvirus expressionsystem; and, wherein the hepatitis B virus structural protein andhepatitis B non-structural proteins assemble into virus-like particles(VLPs) when expressed in the human.
 10. The method of claim 9, whereinthe first nucleic acid sequence is inserted between MVA genes I8R andG1L.
 11. The method of claim 9, wherein the first nucleic acid sequenceis inserted between MVA genes I8R and G1L, and the second nucleic acidsequence is inserted in modified deletion III.
 12. The method of claim9, wherein the promotor is selected from the group consisting of Pm2H5,Psyn II, mH5, P7.5 promoters, or combinations thereof.
 13. The method ofclaim 9, wherein the first nucleic acid sequence comprises SEQ ID NO: 1.14. The method of claim 9, wherein the second nucleic acid sequencecomprises SEQ ID NO: 2 and SEQ ID NO:
 3. 15. The method of claim 9,wherein the first nucleic acid sequence comprises SEQ ID NO: 1 and thesecond nucleic acid sequence comprises SEQ ID NO: 2 and SEQ ID NO: 3.